Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. The HCV genome is a linear, positive-strand RNA molecule of approximately 9,500 nucleotides and encodes a polyprotein of about 3,000 amino acids. Several stretches of amino acids in the HCV polyprotein share significant similarity with flavivirus and pestivirus proteins. Therefore, HCV is considered to be distantly related to these virus groups. The goal of this project is to increase our understanding of the molecular biology of this important human pathogen. Prototype strains of the various genotypes of HCV, including some of those discovered in this laboratory, are being biologically amplified in chimpanzees and further characterized. Pools of virus-contaimng plasma have been packaged and distributed for further characterization and use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Two full-length cDNA clones of HCV (genotypes 1a and 1b) have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. The genotype 1b clone was constructed as a chimera from the infectious cDNA clone of genotype 1a and PCR-amplified sequences from genotype 1b. Four chimpanzees, transfected with genotype 1a or 1b infectious cDNA clones are being followed to determine the natural history of infection. The HVS is monitoring the humoral immune response to these monoclonal viruses and Dr. Frank Chisari (Scripps Institute) is monitoring the cellular immune responses. Upon completion of this study, a detailed analysis of the immune response to experimental HCV infection should be available, a first step toward the development of a rational HCV vaccine. In addition, the availability of infectious cDNA clones of HCV has permitted for the first time a mutational analysis of genomic regions. For example, the 3' non-coding region (NCR) of HCV is complex, consisting of a variable region, a poly U-UC tract and a highly conserved terminal 3' region, but its function is unknown. Individual portions of the 3' NCR have been deleted from the full-length clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Certain regions of the NCR have already been identified as critical for in vivo replication of HCV. By this means, a complete analysis of the 3' NCR region will be completed. Similar analyses of other regions of the HCV genome will be carried out also.